Methods and compositions for treating cardiovascular diseases using fat specific protein 27 (fsp27) compositions

ABSTRACT

FSP27 compositions and methods for treating cardiovascular diseases are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority to U.S. Provisional ApplicationSer. No. 62/703,216 filed Jul. 25, 2018, the entire disclosures of whichare expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was not made with any government support, and thegovernment has no rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Jul. 8, 2019, is named2834_60033_SEQ_LIST_OU18013.txt, and is 19,982 bytes in size.

BACKGROUND OF THE INVENTION

Insulin resistance and cardiovascular diseases are associated disorders,the incidence of which is increasing worldwide. Millions of obese adultsat major risk for diabetes and cardiovascular disease. Despitereductions in preventable global health risks such as tobacco andmalnutrition, obesity has persisted with recent World HealthOrganization data showing that not a single country has been able toreverse obesity trends over the past 33 years and more and moreAmericans are giving up trying to lose weight. Cardiovascular disease isthe leading cause of death in these populations and strategies toreverse cardiometabolic risk are urgently needed. The endothelium playsa critical role in vascular homeostasis and many of its functions aregoverned by the basal and stimulated release of endothelium-derivednitric oxide (NO) that maintains arterial tone, inhibits inflammation,promotes fibrinolysis, and modulates reparative angiogenesis. Animal andclinical studies show that insulin resistance not only perturbsmetabolic pathways in organs such as liver, fat, and muscle, but alsonegatively influences the vasculature. Insulin normally regulates bloodflow through activation of endothelial NO synthase (eNOS) by binding toits IRS-1-linked receptor with subsequent phosphorylation and activationof eNOS via PI3-K/Akt. Defective insulin signaling leads to vascularinflammation, vasoconstriction, plaque progression and ischemia, andendothelium-specific deletion of the insulin receptor in animals causesprofound atherosclerosis.

Further, elevated concentrations of circulating free fatty acids (FFAs)and triglycerides (TGs) are central features of insulin resistance thatare associated with lipotoxicity and systemic endothelial dysfunction.

The lack of therapeutic options and only limited effects of availabledrugs, creates a huge challenge in cardiovascular disease therapy

Thus, there is a great medical need for life-saving treatments for themillions of patients suffering from cardiovascular diseases.

There is no admission that the background art disclosed in this sectionlegally constitutes prior art.

SUMMARY OF THE INVENTION

In a first broad aspect, described herein are uses of FSP27compositions. It is now described herein that the exogenous delivery ofFSP27 is able to rescue FSP27 dysfunction or augment the endogenousfunction of FSP27.

In another broad aspect, described herein are methods of treatment whereadministering exogenous recombinant FSP27 (rFSP27) as a therapeutic forthe treatment of human cardiovascular diseases.

Such uses include, but are not limited to, increasing levels of FSP27 ina subject by administering exogenous recombinant FSP27 (rFSP27).

In certain embodiments, one fragment of FSP27 is comprised of aminoacids 120-140.

Described herein are examples showing the activity of exogenouslyadministered human FSP27 and peptide fragments or analogs in humancardiovascular diseases.

In another broad aspect, described herein are pharmaceuticalcompositions comprising one or more FSP27 medicaments. FSP27 medicamentsmay be administered as a pharmaceutically acceptable salt, or as apegylated composition, or be modified in a pharmaceutically acceptablemanner so as to improve the therapeutic properties. FSP27 medicamentsmay also be administered optionally together with one or more inertcarriers and/or diluents.

The FSP27 medicament is present in an amount sufficient to treat one ormore types of cardiovascular disease.

In another broad aspect, described herein is a method of treating asubject, the method comprising: administering a composition comprising anucleic acid encoding a FSP27 protein or a fragment thereto a subject;wherein, the FSP27 protein has an amino acid sequence having greaterthan 85% homology to at least one of the FSP27 or the FSP27 fragmentsshown.

In certain embodiments, the FSP27 protein has an amino acid sequencehaving greater than about 90% homology to the FSP27 sequences.

In certain embodiments, the FSP27 protein has an amino acid sequencehaving greater than about 95% homology to the FSP27 sequences.

In certain embodiments, the FSP27 protein has an amino acid sequencehaving greater than about 99% homology to the FSP27 sequences.

In certain embodiments, the FSP27 protein is naturally occurring.

In certain embodiments, the FSP27 protein is a recombinant protein.

In certain embodiments, the FSP27 protein comprises a core FSP27 domain,such as amino acids comprising: aa120-239 of FSP27; aa120-230 of FSP27;aa120-210; aa120-140; aa120-220; aa140-210; and/or aa173-220 of FSP27.

In certain embodiments, the subject is a human.

In certain embodiments, the nucleic acid encoding the FSP27 protein isoperably linked to a constitutive transcriptional regulatory sequencecontaining a variety of control elements such as promoters, enhancers,silencers and the like (hereafter collectively called a promoter), anadipocyte-specific promoter, or an inducible promoter.

In certain embodiments, the composition comprises a plasmid, the plasmidcomprising the nucleic acid encoding the FSP27 protein operably linkedto a promoter.

In certain embodiments, the composition comprises a viral vector, theviral vector comprising the nucleic acid encoding the FSP27 proteinoperably linked to a promoter.

Various objects and advantages of this invention will become apparent tothose skilled in the art from the following detailed description of thepreferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the U.S. Patent and Trademark Office upon request andpayment of the necessary fees.

FIG. 1: Schematic illustration of two distinct pathways: left)endothelial cell-specific FSP27 regulation of eNOS signaling andangiogenesis via potential interaction with VEGF; and, right)adipocyte-specific FSP27 regulation of cross-talk with endothelial cellsvia FFA flux as determinants of vascular phenotype.

FIG. 2: FSP27 expression in endothelial cells: FSP27 was distributedthroughout the cytoplasm, and significantly lower in endothelial cellsisolated from visceral vs. subcutaneous depots of obese subjects (n=10,BMI 43±4 kg/m2, *p=0.002; green color=FSP27).

FIG. 3: Vasodilation to insulin improves with rFSP27 (n=10, *p<0.01).FIGS. 4A-4D: Recombinant FSP27 improves insulin signaling in visceraladipose: FIG. 4A—Treatment of visceral fat with rFSP27 decreased basallipolysis. FIG. 4B—Recombinant FSP27 increased Akt and eNOS activity(phosphorylation) in adipose tissue. Quantification ofinsulin-stimulated Akt (FIG. 4C) and eNOS (FIG. 4D) activation in fat.Data are presented as ±SEM. (n=10, *p<0.05).

FIGS. 5A-5B: siRNA-mediated FSP27 knockdown increases lipolysis andimpairs insulin signaling: FIG. 5A—Knockdown of FSP27 in subcutaneousadipose tissue increased rate of glycerol release in the media. Data arepresented as ±SEM. (n=7, *p<0.01); FIG. 5B—siRNA-mediated FSP27depletion decreased Akt and eNOS phosphorylation in response to insulin.

FIGS. 6A-6B: FSP27 enhances in-vitro angiogenic tube formation. Biopsysample of human adipose fat pads showing normal (FIG. 6A) and blunted(FIG. 6B) capillary grow. FSP27 improves blunted angiogenesis.

FIG. 6C: FSP27 enhanced angiogenic tube formation within 6 hrs incultured endothelial cells isolated form the visceral adipose tissue ofobese human subjects (BMI 43±4 kg/m2, n=6, p<0.05).

FIGS. 7A-7B: FSP27 interacts directly with VEGF-A: FIG. 7A: Co-IP ofFSP27 with VEGF-A in endothelial cells; FIG. 7B; IVTT of FSP27 andVEGF-A.

FIGS. 8A-8B: Dual RNA CRISPR/Cas9-mediated FSP27 knockout in HUVECcells: FIG. 8A: Almost complete loss of FSP27 was observed in clones #3, 7 and 15; FIG. 8B; these cells had markedly bluntedinsulin-stimulated AKt phosphorylation.

FIGS. 9A-9C: Endothelial cell immunofluorescence, red color, peNOSsignal: FIGS. 9A-9B display examples of quantitative immunofluorescenceimages, demonstrating normal p-eNOS stimulation to insulin; FIG. 9C,insulin activation of eNOS at Ser1177 is markedly blunted in endothelialcells (EC's) isolated from visceral compared to SC fat.

FIG. 10A: FSP27-KD adipocyte conditioned media added to HUVEC: Theconditioned media was collected at 5th day after the knockdown frommature human subcutaneous white adipocyte.

FIG. 10B-10C: Increased lipolysis in visceral fat is associated withdecreased FSP27 expression: Basal FSP27 was significantly higher insubcutaneous vs. visceral fat depot by mRNA (FIG. 10B) and protein (FIG.10C). Data are presented as ±SEM. (n=13, *p<0.0001 and p<0.001,respectively in paired samples).

FIG. 11A: FSP27-KO mice show blunted capillary formation in adiposetissue. Histochemical staining of capillaries in subcutaneous adiposetissue of mice with isolectin B4.

FIG. 11B: The data represents an average of capillary density in thesubcutaneous adipose tissue of 2 mice per condition and demonstrate a“dose-dependent” loss of capillary network formation with FSPknock-down.

FIG. 12: Generation of ROSA26-FSP27 mice. The 167 bp, band, amplifyingFSP27 exons 3 and 4, confirms successful targeting of FSP27 into theROSA26 locus.

FIGS. 13A-13C: Generation of FSP27tg mice. Schematic representation ofgeneration of: (FIG. 13A) adipose tissue specific (Ad-FSP27tg), and(FIG. 13B) Endothelial specific (E-FSP27tg) human-FSP27 (hFSP27)expressing transgenic mice. (FIG. 13C) Genotyping results of F1 pupscontaining hFSP27 transgene (Ln 1 and 2).

FIG. 14: Schematic illustration of FSP27 fragments/mutants: FSP27(120-239); FSP27 (120-220); FSP27 (120-210); and, FSP27 (140-210).

FIG. 15: Schematic illustration of full length FSP27 showing domainsassociated with lipid droplet dynamics, showing CF4, SEQ ID NO: 4.

FIG. 16: FSP27 sequence is conserved in vertebrates; for example, >90%conserved sequence in FSP27 in: humans (SEQ ID NO: 12); mouse (SEQ IDNO: 13); monkey (SEQ ID NO: 14); dog (SEQ ID NO: 15); cow (SEQ ID NO:16); and, frog (SEQ ID NO: 17).

FIG. 17: Table 1, showing the amino acid sequence detail of the relevantpeptides, listng SEQ ID NOs: 1-12.

FIG. 18: Mice expressing human-FSP27 transgene specifically in theirendothelial cells (E-hFSP27tg) were generating by crossing hFSP27-floxedmice with Tek-cre mice

FIG. 19: Body weight gain in a cohort of floxed and E-hFSP27tg mice onregular diet and high-fat diet.

FIG. 20: Increased oxygen consumption in E-hFSP27tg mice compared toFSP27-floxed (Control) mice. At the age of 4 months these mice were fed60% high-fat diet for 2 months.

FIG. 21: Increased oxygen consumption (CO₂ release) in E-hFSP27tg micecompared to FSP27-floxed (Control) mice. At the age of 4 months thesemice were fed 60% high-fat diet for 2 months.

FIG. 22: Respiratory exchange ratio (RER) in E-hFSP27tg mice compared toFSP27-foxed (Control) mice. At the age of 4 months these mice were fed60% high-fat diet for 2 months.

FIG. 23: Movement (activity) along X axis of E-hFSP27tg mice compared toFSP27-foxed (Control) mice. At the age of 4 months these mice were fed60% high-fat diet for 2 months.

FIG. 24: Movement (activity) along Y axis of E-hFSP27tg mice compared toFSP27-foxed (Control) mice. At the age of 4 months these mice were fed60% high-fat diet for 2 months.

FIG. 25: Movement (activity) along Z-axis of E-hFSP27tg mice compared toFSP27-foxed (Control) mice. E-hFSP27tg mice had more activity/movementalong the Z-axis. At the age of 4 months these mice were fed 60%high-fat diet for 2 months.

FIG. 26: Fasting glucose levels were increased E-hFSP27tg mice comparedto FSP27-foxed (Control) mice. At the age of 4 months these mice werefed 60% high-fat diet (HFD) or regular-chow (RD) diet for 2 months.

FIG. 27: Fasting insulin levels were decreased E-hFSP27tg mice comparedto FSP27-foxed (Control) mice. At the age of 4 months these mice werefed 60% high-fat diet (HFD) for 2 months.

FIG. 28: Glucose tolerance was significantly increased in E-hFSP27tgmice compared to FSP27-floxed (Control) mice. At the age of 4 monthsthese mice were fed 60% high-fat diet (HFD) or regular-chow (RD) dietfor 2 months.

FIG. 29: Insulin tolerance (insulin sensitivity) was significantlyincreased in E-hFSP27tg mice compared to FSP27-floxed (Control) mice. Atthe age of 4 months these mice were fed 60% high-fat diet (HFD) orregular-chow (RD) diet for 2 months.

FIG. 30: Serum Free fatty acid levels in E-hFSP27tg mice compared toFSP27-floxed (Control) mice. At the age of 4 months these mice were fed60% high-fat diet (HFD) or regular-chow (RD) diet for 2 months. Nosignificant change in serum free fatty acid levels was observed.

FIG. 31: Serum triglyceride levels in E-hFSP27tg mice compared toFSP27-floxed (Control) mice. At the age of 4 months these mice were fed60% high-fat diet (HFD) or regular-chow (RD) diet for 2 months. Nosignificant change in serum triglyceride levels was observed.

FIG. 32: Serum adiponectin levels in E-hFSP27tg mice was significantlyincreased compared to FSP27-floxed (Control) mice. At the age of 4months these mice were fed 60% high-fat diet (HFD) where as there was nosignificant change in adiponectin in regular died fed mice.

FIG. 33: Serum leptin levels were decreased in E-hFSP27tg mice comparedto FSP27-foxed (Control) mice. At the age of 4 months these mice werefed 60% high-fat diet (HFD) for 2 months.

FIG. 34: In an in-vitro assay adipose tissue E-hFSP27tg mice showedhigher sprouting indicating increased angiogenesis in E-hFSP27tgcompared to the FSP27-floxed (Control) mice.

FIG. 35: Western blot representing the expression of levels enos, penos,AKT, pAKT, FSP27 and GAPDH in gonadal white adipose tissue of E-hFSP27tgmice and FSP27-floxed (Control) mice. At the age of 4 months these micewere fed 60% high-fat diet (HFD) for 2 months. Results show that AKTphosphorylation (insulin signaling) and eNOS phosphorylation(endothelial function) is improved in E-hFSP27tg mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

Definitions

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

FSP27: Refers to Fat Specific Protein 27, as well as any other acceptednomenclature for the gene in human or other non-human species, includingbut not limited to CIDEC, Cidec, Cide-C, and Cide-3.

FSP27 Compositions/Medicaments: Refers to the FSP27 as shown in theschematic representation of FSP27 fragments, the amino acids, and theamino acid sequences listed in FIGS. 14-17, including any substitutions,deletions, modifications, or mutations thereof.

FSP27 Compositions/Medicaments as contemplated herein may also beprepared as recombinant proteins, including the FSP27 sequences shown inFIG. 16, and in Table 1 in FIG. 17.

The FSP27 protein is encoded by a nucleic acid sequence or gene. As usedherein, a “nucleic acid” or “polynucleotide” includes a nucleic acid, anoligonucleotide, a nucleotide, a polynucleotide, and any fragment orvariant thereof. The nucleic acid or polynucleotide may bedouble-stranded, single-stranded, or triple-stranded DNA or RNA(including cDNA), or a DNA-RNA hybrid of genetic or synthetic origin,wherein the nucleic acid contains any combination ofdeoxyribonucleotides and ribonucleotides and any combination of bases,including, but not limited to, adenine, thymine, cytosine, guanine,uracil, inosine, and xanthine hypoxanthine. The nucleic acid orpolynucleotide may be combined with a carbohydrate, lipid, protein, orother materials. Preferably, the nucleic acid encodes FSP27 protein.

The “complement” of a nucleic acid refers, herein, to a nucleic acidmolecule with sufficient homology to recognize, or which will hybridizeto another nucleic acid under conditions of high stringency.High-stringency conditions are known in the art (see e.g., Maniatis etal., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold SpringHarbor: Cold Spring Harbor Laboratory, 1989) and Ausubel et al., eds.,Current Protocols in Molecular Biology (New York, N.Y.: John Wiley &Sons, Inc., 2001)). Stringent conditions are sequence-dependent, and mayvary depending upon the circumstances. As used herein, the term “cDNA”refers to an isolated DNA polynucleotide or nucleic acid molecule, orany fragment, derivative, or complement thereof. It may bedouble-stranded, single-stranded, or triple-stranded, it may haveoriginated recombinantly or synthetically, and it may represent codingand/or noncoding 5′ and/or 3′ sequences.

In addition, “complementary” means not only those that are completelycomplementary to a region of at least 15 continuous nucleotides, butalso those that have a nucleotide sequence homology of at least 40% incertain instances, 50% in certain instances, 60% in certain instances,70% in certain instances, at least 80%, 90%, and 95% or higher. Thedegree of homology between nucleotide sequences can be determined byvarious methods, including an algorithm, BLAST, etc.

As used herein, nucleic acids and/or nucleic acid sequences are“homologous” when they are derived, naturally or artificially, from acommon ancestral nucleic acid or nucleic acid sequence. Homology isgenerally inferred from sequence identity between two or more nucleicacids or proteins (or sequences thereof). The precise percentage ofidentity between sequences that is useful in establishing homologyvaries with the nucleic acid and protein at issue, but as little as 25%sequence identity is routinely used to establish homology. Higher levelsof sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, or 99% or more can also be used to establish homology. Methodsfor determining sequence similarity percentages (e.g., BLASTN usingdefault parameters) are generally available. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information.

The nucleic acid agent, for example, may be a plasmid. Such a plasmidmay comprise a nucleic acid sequence encoding FSP27, variants orisoforms thereof, although it is to be understood that other types ofnucleic acid agents, such as recombinant viral vectors, may also be usedfor the purposes of the present invention. In one embodiment of thepresent invention, the nucleic acid (e.g., plasmid) encodes at least oneFSP27 variant or isoform.

The term “plasmid”, as used herein, refers generally to circulardouble-stranded DNA, which is not bound to a chromosome. The DNA, forexample, may be a chromosomal or episomal-derived plasmid. The plasmidof the present invention may optionally contain an initiator or promoterof transcription, terminator of transcription, translational controlsequences, and/or a discrete series of restriction-endonucleaserecognition sites, located between the promoter and the terminator. Inthe plasmid, a polynucleotide insert of interest (e.g., one encoding aFSP27-associated protein) should be operatively linked to an appropriatepromoter. The promoter may be its native promoter or a host-derivedpromoter. The promoter may also be a tissue-specific promoter, such asan adipocyte-specific promoter or other tissue-specific promoter. Thepromoter may further be a regulatable promoter, which may be turned offwhen the expression of the gene is no longer desired. Non-limitingexamples of promoters for use in the present invention include the actinpromoter and viral promoters. Other suitable promoters will be known tothe skilled artisan.

Therapeutic: A generic term that includes both diagnosis and treatment.It will be appreciated that in these methods the “therapy” may be anytherapy for treating a disease including, but not limited to,pharmaceutical compositions, gene therapy and biologic therapy such asthe administering of antibodies and chemokines. Thus, the methodsdescribed herein may be used to evaluate a patient or subject before,during and after therapy, for example, to evaluate the reduction indisease state.

Adjunctive therapy: A treatment used in combination with a primarytreatment to improve the effects of the primary treatment.

Clinical outcome: Refers to the health status of a patient followingtreatment for a disease or disorder or in the absence of treatment.Clinical outcomes include, but are not limited to, an increase in thelength of time until death, a decrease in the length of time untildeath, an increase in the chance of survival, an increase in the risk ofdeath, survival, disease-free survival, chronic disease, metastasis,advanced or aggressive disease, disease recurrence, death, and favorableor poor response to therapy.

Decrease in survival: As used herein, “decrease in survival” refers to adecrease in the length of time before death of a patient, or an increasein the risk of death for the patient.

Patient: As used herein, the term “patient” includes human and non-humananimals. The preferred patient for treatment is a human. “Patient,”“individual” and “subject” are used interchangeably herein.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop.“Ameliorating” refers to the reduction in the number or severity ofsigns or symptoms of a disease.

Poor prognosis: Generally refers to a decrease in survival, or in otherwords, an increase in risk of death or a decrease in the time untildeath. Poor prognosis can also refer to an increase in severity of thedisease.

Screening: As used herein, “screening” refers to the process used toevaluate and identify candidate agents that affect such disease.

Comprising, comprises and comprised of: As used herein are synonymouswith “including”, “includes” or “containing”, “contains”, and areinclusive or open-ended and do not exclude additional, non-recitedmembers, elements or method steps. The terms “comprising”, “comprises”and “comprised of” also include the term “consisting of”.

About: As used herein when referring to a measurable value such as aparameter, an amount, a temporal duration, and the like, is meant toencompass variations of +/−10% or less, preferably +/−5% or less, morepreferably +/−1% or less, and still more preferably +/−0.1% or less ofand from the specified value, insofar such variations are appropriate toperform in the disclosed invention. It is to be understood that thevalue to which the modifier “about” refers is itself also specifically,and preferably, disclosed.

And/or: When used in a list of two or more items, means that any one ofthe listed items can be employed by itself or any combination of two ormore of the listed items can be employed. For example, if a list isdescribed as comprising group A, B, and/or C, the list can comprise Aalone; B alone; C alone; A and B in combination; A and C in combination,B and C in combination; or A, B, and C in combination.

EXAMPLES

Certain embodiments of the present invention are defined in the Examplesherein. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

Fat Specific Protein (FSP27), also known as cell death-inducingDFFA-like effector (CIDEC in humans and Cidec in mice; also abbreviatedCide-c or Cide-3) is a member of the cell death-inducing DNAfragmentation factor-like effector family—a group of genes that play animportant role in apoptosis. The invention described herein identifiesan additional, novel role of FSP27 as a therapeutic target for treatingcardiovascular disease.

It is now shown herein that FSP27 protein increases eNOS activity (whichis a marker of vascular function).

It is also now shown herein that FSP 27 is abundantly expressed invascular endothelial cells in intracellular network formationscompletely different than in adipocytes, and is down-regulatedparticularly in association with visceral obesity. It is now believedthat perturbations in FSP27 promote conditions that elevate FFAs whichare implicated in insulin resistance and endothelial dysfunction. Whilelipid storage or breakdown is generally not viewed as a primary functionof vascular endothelial cells, it is now believed that FSP27 governscellular responses by mechanisms beyond regulation of lipid metabolism,such as by modulating signal transduction or functioning as a chaperoneco-receptor for other proteins. While no wishing to be bound by theory,it is also now believed that FSP27 serves as a critical regulator ofarteriolar vasodilator capacity and angiogenesis which are pivotal inmechanisms of atherosclerosis and ischemic cardiovascular disease.

The domains and/or sequences of the FSP27 protein and rFSP27 are usefulas peptides to protect epithelial cells against insulin resistance andcardiovascular disease.

FIG. 1 is a schematic illustration of FSP27 and its intricate signalingsystem which is a previously unrecognized but functionally significantmodulator of vascular phenotype.

FSP27 is highly expressed in human endothelial cells derived from theadipose tissue microvasculature. As shown in FIG. 2, usingimmunohistochemistry and confocal imaging, FSP27 protein was clearlydetectable and significantly lower in endothelial cells isolated fromvisceral compared to subcutaneous depots of obese subjects. This is asurprising finding because endothelial cells are generally notconsidered primary storage sites for lipid metabolism. It is nowbelieved that FSP27 has an alternative function in vascular cells—suchas modulation of signal transduction. Also, see FIGS. 10B-10C which showendothelium-specific FSP27 expression, which was knocked-out usingCRISPR/Cas9, thereby showing FSP27′s cell autonomous role.

It was also determined whether there are any direct effects of FSP27 onvascular responses by examining the effects of human recombinant protein(rFSP27). The primary readout for vascular function involved two keyphysiological functions of blood vessels: A) endothelium-dependentvasodilation to insulin (as an index of vascular insulin resistance) andB) angiogenic growth capacity (a marker of capillary regenerativepotential). Both of these parameters become dysfunctional under diseaseconditions and contribute to mechanisms of adipose tissue dysfunctionlocally, and atherosclerosis and ischemic injury systemically.

The endothelium-dependent arteriolar vasodilator function of themicrovasculature within human adipose tissue was examined usingvideo-microscopy. As shown in FIG. 3, time-response to insulin-mediated,nitric-oxide mediated vasodilation was severely blunted (red plot) inthe visceral adipose tissue microvasculature. Responses tonon-endothelium-dependent vasodilation to papavarine were intactindicating a functional defect specifically at the level of theendothelium. Treatment with rFSP27 (blue plot) significantly improvedvasomotor function (n=10, p<0.01) and the positive response was nearlyfully abolished by eNOS inhibitor N^({acute over (ω)})-nitro-L-argininemethyl ester (L-NAME, 100 μM) showing that the beneficial mechanisms wasrelated primarily to improved NO bioaction. To confirm, whole visceralfat was exposed to rFSP27 and examined eNOS phosphorylation (p-eNOS) atserine 1177, the commonly reported major index of eNOS stimulatoryactivation in endothelial cells.

As shown in FIGS. 4A-4D, rFSP27 reduced basal lipolysis (FIG. 4A) andmarkedly improved insulin-mediated Akt and eNOSactivation/phosphorylation (FIGS. 4B-4D) in the visceral fat of obesehumans. In particular, FIG. 4D shows the insulin-mediated activation ofeNOS and AKT in response to recombinant human FSP27 in the visceraldepot, and the quantification of percent change in insulin-mediatedactivation of eNOS at baseline and after 24 hours of treatment withrFSP27 in the visceral depot. (n=10, p<0.05). Data are presented asarbitrary units (au) and as mean±SEM. *P≤0.05.

These findings strongly complement the intact vessels physiologicalstudies shown in FIG. 3. Conversely, siRNA methods were used to silenceFSP27 (˜70% silencing action) in human fat which had opposite effect torFSP27, and increased lipolysis in parallel with impairedinsulin-mediated Akt and eNOS phosphorylation in SC fat (FIGS. 5A-5B).

It was also determined whether FSP27 influences broader functions ofblood vessels and specifically focused on angiogenesis. Adipose tissueangiogenesis was examined using ex-vivo Matrigel based assays thatexamine capillary sprout growth in adipose tissue specimens. Defectiveangiogenesis in the adipose tissue has been linked to insulinresistance, however mechanisms are unknown. Also, anti-angiogenicmediators produced in human fat are detectable in circulating blood andlikely have systemic effects.

Illustrations of “preserved” and “blunted/abnormal” angiogenic growth inhuman fat pads are shown in FIGS. 6A-6B. A complimentary method ofangiogenic assessment involves a tube formation assay that quantifiesthe capacity of endothelial cells plated onto gelled basement matrix toorganize and coalesce into capillary-like structures with a rudimentarylumen within hours. As shown in FIG. 6C, isolated primary endothelialcells from living obese subjects from the visceral depot exhibited weakvasculogenic behavior (control), whereas rFSP27 significantly improvedchannel formation at 6 hrs (n=6, *p<0.05).

This evidence is striking because FSP27 was initially believed to beexclusively involved in the regulation of adipocyte lipid metabolism.However, these data show that there is a key role and functionalsignificance of FSP27 as a critical endogenous modulator of vascularfunction in human obesity.

To determine the role of VEGF-A as a candidate mediator of FSP27 action,interaction between these two proteins was examined, a strongco-immunoprecipitation was observed (FIG. 7A). An in vitrotranscription-translation coupled co-IP immunoblot assay (IVTT) wasconducted which confirmed a direct interaction of FSP27 with VEGF (FIG.7B). It is now believed that FSP27 functions both at the nexus ofextracellular signaling via lipotoxicity, and intracellular pathways byinterfering with cell-autonomous endothelial responses.

Vascular cell FSP27 regulates angiogenesis through the VEGF-Akt-eNOSpathway.

To determine the role of FSP27 in endothelial function, gain-and-loss offunction are performed in cultured endothelial cells lines as genemanipulation can be readily performed in these cultured systems. anFSP27 knockout system in human umbilical vein endothelial cell (HUVEC)lines by a dual RNA mediated CRISPR/Cas9 method is used, and achievednearly full loss of FSP27 expression as shown in (FIG. 8A). Thisproduced an approximately 90% decrease in insulin-stimulated Aktphosphorylation (FIG. 8B) showing that FSP27 regulates eNOS activationvia Akt signaling.

Also, arterial cell lines (HAECs) and primary endothelial cells that areobtained from different fat depots of human subjects are also examined(as in FIGS. 6A-6C).

As shown in FIG. 1, and data in FIGS. 7A-7B, FSP27-VEGF has autocrineand/or paracrine effects on eNOS activity and angiogenesis.

Angiogenic regulation of FSP27 is determined by blocking intracellularVEGF (autocrine pathway), and extracellular VEGF and VEGFR2 (paracrinepathway). In these experiments, VEGF or VEGFR2 is depleted using RNAi.Further, in order to confirm the role of FSP27-VEGF interactions invascular modeling, FSP27^(−/−) HAEC cells and use recombinant VEGF areused to determine whethe FSP27 is required in the process of eNOSactivation and tube formation.

Role of endothelial-specific FSP27 in insulin signaling in endothelialcells.

The activation of eNOS and angiogenesis is dependent upon insulinsignaling. The data in FIGS. 4A-D and FIGS. 5A-5B clearly show theeffect of FSP27 on eNOS activity and Akt phosphorylation in humanadipose tissue. However, the effect of FSP27 on Akt activity could bemainly due to the Akt in adipocytes.

To determine the specific role of FSP27 on insulin signaling via Akt inendothelial cells, the role of FSP27 is investigated in basal andinsulin-stimulated Akt activation in HAEC cells in the presence orabsence of FSP27, monitored by Akt phosphorylation at Ser⁴⁷³ and Ser³⁰⁸sites.

The protocol includes examining eNOS phosphorylation at serine1177(p-eNOS), the commonly reported major index of eNOS stimulatoryactivation in endothelial cells.

FIGS. 9A-9B display examples of quantitative immunofluorescence images,demonstrating normal p-eNOS stimulation to insulin, where the FIG. 9Adepicts ambient basal conditions, and FIG. 9B panel shows potent insulinstimulation of p-eNOS at Ser1177 (depicted by intensification of the redsignal). DAPI nuclear stain (blue) and von Willebrand factor (vWF,green) are standard markers used to identify endothelial cells. As shownFIG. 9C, insulin activation of eNOS at Ser1177 is markedly blunted inendothelial cells (EC's) isolated from visceral compared to SC fat.

As shown in FIG. 9C, the data show that insulin activation of eNOS atSer1177 is markedly blunted in endothelial cells (EC's) isolated fromvisceral compared to subcutaneous fat of obese subjects (**p<0.01, n=7,%change).

Role of adipocyte-specific FSP27 in cross-talk between adipocytes andendothelial cells.

FSP27 depletion in human primary adipocytes increases lipolysis andaugmenting FFAs release in the media. In turn, elevated FFAs in obesityinhibits insulin-stimulated eNOS activation and promotes endothelialdysfunction.

FSP27-KD adipocyte conditioned media added to HUVEC: The conditionedmedia was collected at 5th day after the knockdown from mature humansubcutaneous white adipocyte. This media was added to the HUVEC cells.Insulin-stimulated Akt phosphorylation was measured in HUVEC cells after2days of incubation with the conditioned media. FIG. 10A is arepresentative of three different sets of experiments performed with twodifferent concentrations of FSP27 siRNA (50 nM,75 nM).

While not wishing to be bound by theory, it is now believed thatlipolytic flux of FFAs regulated by FSP27 in adipocytes affect eNOSsignaling and angiogenesis in endothelial cells in a paracrine manner,potentially through FFA. FIGS. 10B-10C show decreased FSP27 expressionin the visceral adipose tissue of obese humans that is associated withincreased lipolysis.

FSP27 over-expression protects against vascular dysfunction.

The role of FSP27 is examined in protection against obesity inducedvascular dysfunction using adipose-specific and endothelial-specifichuman FSP27-overexpressing transgenic mice. Until now, the vascularpathophysiological importance of FSP27 and the associated signaling inthese constructs have never been examined.

FSP27-KO mice showed blunted capillary formation in epididymal adiposetissue. There is marked impairment in capillary formation (FIG. 11A andFIG. 11B), showing a role for FSP27 in regulating endothelial functionand vascular proliferation. The capillary density was proportional toFSP27 expression, as heterozygous knockout mice (FSP27^(+/−)) showed anintermediate phenotype compared to the wild-types and full FSP27^(−/−)mice. While not wishing to be bound by theory, it is now believed thatadipose-specific FSP27 overexpression protects from HFD-inducedlipolysis, insulin resistance and endothelial dysfunction. Miceover-expressing human FSP27 specifically in fat or endothelial cells areused to characterize the cell-autonomous function(s) of FSP27 inregulating insulin signaling, eNOS activation, angiogenesis,vasodilation, and metabolic phenotype.

Generation of adipocyte-specific as well as endothelial-specific humanFSP27-overexpressing transgenic mice.

FSP27-overexpression is vasculo-protective. FSP27 has been cloned inROSA26-CMV-loxSTOPlox vector and generated mice which conditionallyover-express FSP27 (FIG. 12).

The mice were crossed with Adipoq-cre mice to specifically over-expressFSP27 in adipose tissue, (Ad-FSP27tg). First generation Ad-FSP27tg miceare shown in (FIG. 13A). To generate endothelial-specific FSP27tg mice,the conditionally overexpressed FSP27 mice (FIG. 12) are crossed withB6.Cg-Tg(Tek-cre)1Ywa/J mice to specifically overexpress FSP27 inendothelial cells (E-FSP27tg mice) (FIG. 13B). FIG. 13C shows thegenotyping results of F1 pups containing hFSP27 transgene (Ln 1 and 2).

Measurement of vascular parameters in Ad-FSP27tg and E-FSP27tg mice.Human data showed that arteriolar vasodilator and angiogenic function ofthe microvasculature in human adipose tissue was enhanced upon treatmentwith recombinant FSP27 (see FIG. 3, FIG. 6). Thus, again, while notwishing to be bound by theory, it is now believed that overexpression ofFSP27 in adipose tissue and/or endothelial cells protects mice fromhigh-fat diet induced endothelial dysfunction.

To confirm that these animal models show the cell-specific importance ofFSP27 over-expression, and their respective vascular and metabolicphenotypes, the insulin responsiveness of adipose tissue is to beassessed by collecting tissues 10 minutes after intraperitoneal deliveryof 0.75 U/kg of insulin. Akt and eNOS phosphorylation are to beevaluated by Western Blot and densitometric quantification of thepAkt(S473)/Akt and peNOS(S1179)/eNOS ratios. Also, phosphorylation ofInsRβ, Aktl, ERK1/2, foxo1, and eNOS (by Western analysis) in aorta andfemoral arteries is to be examined, since endothelial cells constituteapprox. 50% of cellular populations in this tissue.

Further, endothelial cells will be isolated from the adipose tissues oftest mice and WT mice by FACS sorting and analyzed forinsulin-stimulated Akt phosphorylation and eNOS phosphorylation atbaseline and in the presence of exogenous recombinant FSP27.

Endothelial function/dysfunction in mouse aortic rings harvested fromthe different experimental groups of mice (WT vs. transgenic mice undernormal chow vs. high fat diet) are also to be evaluated.Insulin-stimulated vasodilation is to be assessed by standard methodsthat are similar to the studies performed on human vessels harvestedfrom fat (see FIG. 3).

For comparison and further characterization, vessel contractile andrelaxation responses to phenylephrine and insulin, respectively, arealso assessed. Capillary density is to be examined by histochemicalstaining of adipose tissue capillaries using isolectin B4, andquantified as shown in FIG. 12. Angiogenic capacity is to be assessedusing fat-pad sprouting assays and tube formation as depicted in FIG. 6.

FIG. 14 is a schematic illustration of FSP27 fragments/mutants: FSP27(120-239); FSP27 (120-220); FSP27 (120-210); and, FSP27 (140-210).

FIG. 15 is a schematic illustration of full length FSP27 showing domainsassociated with lipid droplet dynamics, and the segment that has shownmaximum efficacy towards cardiovascular disease effect in cells.

FIG. 16: FSP27 sequence is conserved in vertebrates; for example, >90%conserved sequence in FSP27 in: humans (SEQ ID NO: 12); mouse (SEQ IDNO: 13); monkey (SEQ ID NO: 14); dog (SEQ ID NO: 15); cow (SEQ ID NO:16); and, frog (SEQ ID NO: 17).

FIG. 17: Table 1, showing the amino acid sequence detail of the relevantpeptides, listng SEQ ID NOs: 1-12.

Adipocyte-specific lipid-droplet associated protein, FSP27, is alsoexpressed in endothelial cells.

A mouse model was generated that expresses a single allele of FSP27transgene specifically in the endothelial cells (E-hFSP27tg) (See FIG.18). On regular chow and high-fat diets, these mice gain weight similarto the control mice (floxed-wild type) (See FIG. 19).

Even after feeding high-fat diet, their metabolic profile, like VO₂,VCO_(2,) and RER is better compared to the control mice. FIGS. 20, 21,22 show that the model mice have higher fatty acid oxidation in themuscle.

The model mice also show higher activity (FIGS. 23, 24, 25).

Both regular-chow and high-fat-fed E-hFSP27 mice have lower fastingblood glucose compared to the controls (FIG. 26), and E-hFSP27tg miceshowed lower fasting insulin levels showing that they are protectedagainst hyperinsulinemia (FIG. 27).

The model mice are protected against high-fat diet induced insulinresistance as shown by glucose tests (FIG. 28) insulin tolerance tests(FIG. 29).

The circulatory fatty acids and triglycerides are normal (FIGS. 30-31).Serum adiponectin levels in E-hFSP27tg mice were higher (FIG. 32)whereas leptin levels were lower (FIG. 33) than the control mice.

Angiogenesis, insulin sensitivity, and eNOS activation in the adiposetissue of control vs transgenic mice were examined to determine themechanism of action. eNOS activation is associated with angiogenesis andvasodilation of blood vessels, which is protective againstcardiovascular disease. The results showed higher capillary sprouting(FIG. 34) in adipose tissue of E-hFSP27tg mice associated with higherinsulin-stimulated AKT and eNOS phosphorylation (FIG. 35).

Overall, the results show that the transgenic mice expressionhuman-FSP27 in endothelial cells are protected against insulinresistance and show higher systemic metabolic health.

Other Examples

Pharmaceutical Compositions

A pharmaceutical composition as described herein may be formulated withany pharmaceutically acceptable excipients, diluents, or carriers. Acomposition disclosed herein may comprise different types of carriersdepending on whether it is to be administered in solid, liquid, oraerosol form, and whether it needs to be sterile for such routes ofadministration as injection. Compositions disclosed herein can beadministered in a suitable manner, including, but not limited totopically (i.e., transdermal), subcutaneously, by localized perfusionbathing target cells directly, via a lavage, in creams, in lipidcompositions (e.g., liposomes), formulated as elixirs or solutions forconvenient topical administration, formulated as sustained releasedosage forms, or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 2003, incorporated herein byreference).

The compositions provided herein are useful for treating animals, suchas humans. A method of treating a human patient according to the presentdisclosure includes the administration of a composition, as describedherein.

The phrases “pharmaceutical” or “pharmacologically acceptable” refer tomolecular entities and compositions that produce no adverse, allergic,or other untoward reaction when administered to an animal, such as, forexample, a human. A carrier or diluent may be a solid, semi-solid, orliquid material which serves as a vehicle, excipient, or medium for theactive therapeutic substance. Some examples of the diluents or carrierswhich may be employed in the pharmaceutical compositions of the presentdisclosure are lactose, dextrose, sucrose, sorbitol, mannitol, propyleneglycol, liquid paraffin, white soft paraffin, kaolin, fumed silicondioxide, microcrystalline cellulose, calcium silicate, silica,polyvinylpyrrolidone, cetostearyl alcohol, starch, modified starches,gum acacia, calcium phosphate, cocoa butter, ethoxylated esters, oil oftheobroma, arachis oil, alginates, tragacanth, gelatin, syrup, methylcellulose, polyoxyethylene sorbitan monolaurate, ethyl lactate, methyland propyl hydroxybenzoate, sorbitan trioleate, sorbitan sesquioleateand oleyl alcohol, and propellants such as trichloromonofluoromethane,dichlorodifluoromethane, and dichlorotetrafluoroethane.

The phrase “chemotherapeutic agent” refers to a therapeutic agent knownto be used in treating a subject that has been diagnosed withcardiovascular disease. Some examples of general classes ofchemotherapeutic agents of the present disclosure include alkylatingagents, anthracyclines, cytoskeletal disruptors, epothilones, histonedeacetylase inhibitors, inhibitors of topoisomerase I and II, kinaseinhibitors, nucleotide analogs and precursor analogs, peptideantibiotics, platinum-based agents, retinoids, and vina alkaloids andderivatives. Of these general classes, specific examples include but arenot limited to doxorubicin (Adriamycin), sorafenib tosylate, cisplatin,paclitaxel, gemcitabine, vemurafenib, dabrafenib, linsitinib,crizotinib, and cabozantinib.

Solutions of the compositions disclosed herein as free bases orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof, and in oils. Under ordinary conditions of storage and use,these preparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile solutions or dispersions. Incertain cases, the form should be sterile and should be fluid to theextent that easy injectability exists. It should be stable under theconditions of manufacture and storage and may optionally be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, a polyol (i.e., glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and/or vegetable oils. The prevention of the action of microorganismscan be brought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it may be preferable to include isotonicagents, such as, but not limited to, sugars or sodium chloride.

Pharmaceutical compositions for topical administration may include thecompositions formulated for a medicated application such as an ointment,paste, cream, or powder. Ointments include all oleaginous, adsorption,emulsion, and water-soluble based compositions for topical application,while creams and lotions are those compositions that include an emulsionbase only. Topically administered medications may contain a penetrationenhancer to facilitate adsorption of the active ingredients through theskin. Suitable penetration enhancers include glycerin, alcohols, alkylmethyl sulfoxides, pyrrolidones and luarocapram. Possible bases forcompositions for topical application include polyethylene glycol,lanolin, cold cream, and petrolatum as well as any other suitableabsorption, emulsion, or water-soluble ointment base. Topicalpreparations may also include emulsifiers, gelling agents, andantimicrobial preservatives as necessary to preserve the composition andprovide for a homogenous mixture. Transdermal administration of thecompositions may also comprise the use of a “patch.” For example, thepatch may supply one or more compositions at a predetermined rate and ina continuous manner over a fixed time-period.

It is further envisioned the compositions disclosed herein may bedelivered via an aerosol. The term aerosol refers to a colloidal systemof finely divided solid or liquid particles dispersed in a liquefied orpressurized gas propellant. The typical aerosol comprises a suspensionof active ingredients in liquid propellant or a mixture of liquidpropellant and a suitable solvent. Suitable propellants includehydrocarbons and hydrocarbon ethers. Suitable containers can varyaccording to the pressure requirements of the propellant. Administrationof the aerosol can vary according to subject's age, weight, and theseverity and response of the symptoms.

Dosage

The actual dosage amount of a composition disclosed herein administeredto an animal or human patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient, and the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The compounds of the present disclosure are generally effectiveover a wide dosage range. The practitioner responsible foradministration can, in any event, determine the concentration of activeingredient(s) in a composition and appropriate dose(s) for theindividual subject.

Naturally, the amount of active compound(s) in each therapeuticallyuseful composition may be prepared in such a way that a suitable dosagecan be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations can be contemplated by those preparing suchpharmaceutical formulations, and as such, a variety of dosages andtreatment regimens may be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. hi non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above. The dosages can depend on many factors, and canin any event be determined by a suitable practitioner. Therefore, thedosages described herein are not intended to be limiting.

In some embodiments, the compositions further include an additionalactive ingredient. The preparation of a pharmaceutical composition thatcontains at least one compound or additional active ingredient can beknown to those of skill in the art in light of the present disclosure,as exemplified by Remington's Pharmaceutical Sciences, 2003,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it can be understood that preparations should meetsterility, pyrogenicity, and general safety and purity standards asrequired by the FDA Office of Biological Standards.

Packaging of the Composition

After formulation, the composition is packaged in a manner suitable fordelivery and use by an end user. In one embodiment, the composition isplaced into an appropriate dispenser and shipped to the end user.Examples of final container may include a pump bottle, squeeze bottle,jar, tube, capsule or vial.

The compositions and methods described herein can be embodied as partsof a kit or kits. A non-limiting example of such a kit comprises theingredients for preparing a composition, where the containers may or maynot be present in a combined configuration. In certain embodiments, thekits further comprise a means for administering the composition, such asa topical applicator, or a syringe. The kits may further includeinstructions for using the components of the kit to practice the subjectmethods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be present in the kits as a package insert or in thelabeling of the container of the kit or components thereof. In otherembodiments, the instructions are present as an electronic storage datafile present on a suitable computer readable storage medium, such as aflash drive, CD-ROM, or diskette. In other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, such as via the internet, areprovided. An example of this embodiment is a kit that includes a webaddress where the instructions can be viewed and/or from which theinstructions can be downloaded. As with the instructions, this means forobtaining the instructions is recorded on a suitable substrate.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof.

Therefore, it is intended that the invention not be limited to theparticular embodiment disclosed herein contemplated for carrying outthis invention, but that the invention will include all embodimentsfalling within the scope of the claims.

1. A method for inhibiting cardiovascular disease in a subject,comprising, administering an FSP27 medicament or a pharmaceuticallyacceptable composition thereof, in an amount to treat cardiovasculardisease.
 2. The method according to claim 1, wherein the cardiovasculardisease is selected from one or more of the following: cardiovascularconditions: ischemic heart disease, coronary artery disease (CAD),angina, infarction, coronary syndrome, peripheral artery disease (PAD),cerebrovascular disease, stroke, congestive heart failure, systolic ordiastolic cardiomyopathy, insulin resistance, vascular spasm,vasospastic angina, cardiac arrhythmia, impaired angiogenesis, reducedvascular growth.
 3. The method of claim 1, wherein the FSP27 medicamentcomprises full length FSP27 [SEQ ID NO: 12], or peptide fragmentsthereof.
 4. The method of claim 3, wherein the FSP27 fragment is one ormore of SEQ ID NOS: 1-11, or variants or isoforms thereof.
 5. The methodof claim 3, wherein the FSP27 fragment comprises Cf4, having SEQ IDNO:4.
 6. The method of claim 1, wherein the FSP27 medicament isco-administered with at least one additional therapeutic agent.
 7. Amethod of treating cardiovascular disease in a subject, the methodcomprising: administering a composition comprising a nucleic acidencoding a FSP27 protein to a subject; wherein the FSP27 protein has anamino acid sequence having greater than about 85% homology to at leastone of the FSP27 sequences shown in FIG. 16 or fragments shown in Table1 in FIG.
 17. 8. (canceled)
 9. An FSP27 medicament, or a pharmaceuticalcomposition thereof, comprising a FSP27 fragment, as shown in Table 1 inFIG. 17, or variants or isoforms thereof.
 10. The pharmaceuticalcomposition of claim 9 further comprising one or more FSP27 medicaments,or one or more pharmaceutically acceptable modifications of FSP27thereof, optionally together with one or more inert carriers and/ordiluents, the FSP27 medicament being present in an amount sufficient totreat one or more cardiovascular diseases.
 11. The composition of claim9, wherein the FSP27 protein variant or isoform thereof has an aminoacid sequence having greater than about 85% homology to the FSP27sequences.
 12. The composition of claim 9, wherein the FSP27 proteinvariant or isoforms thereof has an amino acid sequence having greaterthan about 90% homology to the FSP27 sequences.
 13. The composition ofclaim 9, wherein the FSP27 protein variant or isoforms thereof has anamino acid sequence having greater than about 95% homology to the FSP27sequences.
 14. The composition of claim 9, wherein the FSP27 proteinvariant or isoforms thereof has an amino acid sequence having greaterthan about 99% homology to the FSP27 sequences.
 15. The composition ofclaim 9, wherein the FSP27 protein is naturally occurring.
 16. Thecomposition of claim 9, wherein the FSP27 protein is a recombinantprotein.
 17. The composition of claim 9, wherein the FSP27 proteincomprises a core FSP27 domain comprising one of: aa120-239; aa120-230;aa120-210; aa120-140; aa120-220; aa140-210; and/or aa173-220.
 18. Thecomposition of claim 9, wherein the FSP27 protein is a human protein.19. The composition of claim 9, wherein the subject is a human.
 20. Thecomposition of claim 9, wherein the subject experiences reducedcardiovascular disease.
 21. The composition of claim 9, wherein thenucleic acid encoding the FSP27 protein is operably linked to apromoter.
 22. The composition of claim 9, wherein the compositioncomprises a plasmid, the plasmid comprising the nucleic acid encodingthe FSP27 protein operably linked to a promoter.
 23. The composition ofclaim 9, wherein the composition comprises a viral vector, the viralvector comprising the nucleic acid encoding the FSP27 protein operablylinked to a promoter.